The goal of this proposal is to study the composition and structure of the multidrug resistance pump EmirAB, with the long-term aim of understanding its mechanism. Identifying the binding site of th small soluble EmirR will serve as an important model in aiding our long-term goal of understanding the mechanism of drug discrimination by the large membrane MDR. EmirR is expressed at a high level and might participate more directly in the faction of the EmrAB pump by sequestering its substrates. Envelope MDRs play a significant role in resistance of pathogenic gram-negative bacteria, underscoring the importance of understanding their mechanism of action in order to design effective antimicrobials. Specific Aims: 1. Interaction between components of the pump. EmrB is an integral membrane protein related to such extrusion pumps as the tetracycline/H+- antiporters. EmrA belongs to a family of 'membrane fusion proteins" that connect the outer and inner membranes of gram-negative bacteria. The link would allow the pump to extrude toxins across the outer membrane which is a good barrier for bulky hydrophobic substances. Interaction between EmirB and EmirA will be studied by crosslinking and identification of interacting species with Western blotting. A pump extruding a ligand across the outer membrane would need to be docked to a porin. A specialized porin TolC is part of peptide extrusion pumps that contain an EmirA-like fusion protein. Our data indicate the TolC is required for the EmirAB function as well. Crosslinking will be used to identify possible interaction between TolC and EmirA/EmrB. 2. Cellular localization of the pump, structure/function. Immunogold labeling and electron microscopy will be used to localize EmirA, EmrB and TolC int he cell (in collaboration with Dr. Manfred Bayer). It is possible that the MDR will be located in membrane adhesion zones, where athe outer and inner membranes form a contact. In an independent approach, the membrane contact fraction will be isolated physically and assayed for the presence of the pump components. Active transport measurements will be performed to learn whether EmirAB indeed transports ligands across the outer membrane barrier. 3. Detailed membrane topography of EmirA and EmirB. Construction of random fusions with beta-lactamase will be used to locate both cytoplasmic and periplasmic domains of EmirA and EmirB. A periplasmic domain(s) of EmirB that is a good candidate for EmrA interaction will be localized in this manner. Domains of EmirB that are essential for transport will be identified by measuring the transport activity of different EmirB-beta- lactamase fusions. 4. EmrR and drug binding. Gel mobility shift analysis and equilibrium binding studies with EmirR and ligands will be performed emrR is the first gene of the emrRAB operon, it ends with a terminator, and is apparently expressed at high levels. Its suggested that Emir has a more direct role in resistance, acting as a drug sequestering mechanism. This hypothesis will be tested by studying resistance in delta emrAB cells upon induction of EmrR expression. EmrR might be an ideal model to study the mechanism by which MDRs discriminate between drugs and cell components. The structure of this 19kD soluble protein will be studied by NMR spectroscopy with the aim of identifying the ligand binding site. Purified EmrR will also be used to obtain crystals for X-ray diffraction analysis (by Dr. Gregory Petsko).